Vaccines for, e.g., subcutaneous injection may be prepared by mixing an aqueous solution of an antigen and a solid phase carrier, e.g. aluminum hydroxide gel, to produce a mixture, wherein at least a part of the antigen is adsorbed to the solid phase and part of or none of the antigen is in the liquid phase. The solid phase carrier may serve as an adjuvant, i.e. it potentiates the immune response of the antigen, although the mechanism of the potentiation is not always fully understood. Also, the mechanism and nature of the adsorption of the antigen to the solid phase carrier is not always fully understood and may depend strongly on the type of antigen involved. Theoretically, however, the adsorption to aluminum hydroxide gels partly involves electrostatic forces. For proteins, it is believed that the phosphate groups of phosphorylated proteins also interact with the aluminum hydroxide gel and possibly to some extent replaces the hydroxide groups in the gel structure.
Consequently, the degree of adsorption varies with the nature of the specific protein in question. Also, in the case of an antigen in the form of an extract of a biological material, e.g. an extract of grass pollen allergens, the extract contains a number of different ions and molecules, which potentially interferes with the bonding of the allergens to the solid phase carrier.
The immunological activity may be measured in an in vivo method involving the administration of the vaccine to a test animal to raise antibodies to the antigen, collecting a biological sample and assaying the sample to measure the amount of antibodies raised. The allergenic activity and the potential for inducing allergic reactions may be tested by intradermal injection in sensitized animals, and by measurement of the extent of the wheel and flare reactions (Kildsgaard et al., Assessment of the in vivo allergenic potency of new allergy vaccines by intradermal testing in sensitized mice, Clinical Immunology and Allergy in Medicine, Proceedings of the 21st EAACI Congress 2002, Naples, Italy). However, such in vivo methods are laborious and time-consuming, and they necessitate the use of test animals, which is undesirable.
Russian Patent No. SU-A-1 746 318 discloses a method of quantitative determination of antigen in tick-borne encephalitis vaccine preparation, wherein the antigen is adsorbed on aluminum hydroxide, the method comprising reacting the vaccine preparation with an excess amount of specific antibodies in phosphate buffer, followed by immunoenzymatic determination of the amount of antibodies in the supernatant. A calibration graph is used to obtain quantitative results.
Chang et al. (Vaccine 19 (2001) 2884-2889) discloses a study examining the degree of lysozyme adsorption to aluminum hydroxide gel in the vaccine and in interstitial fluid and its effect on the immune response in rabbits. Vaccines were pre-treated with phosphate anion to produce vaccines having degrees of adsorption ranging from 3 to 90%. It was found that the degree of adsorption of vaccines exhibiting 3, 35 or 85% adsorption changed to 40% within one hour of mixing with interstitial fluid to simulate subcutaneous administration. In accordance with this, the anti-lysozyme antibody response was the same for vaccines having different degrees of adsorption.
Shi et al. (Vaccine 20 (2002) 80-85) discloses a study of the ability of interstitial fluid to change the degree of adsorption of ovalbumin to aluminum hydroxide adjuvant and lysozyme to aluminum phosphate adjuvant. Ovalbumin and lysozyme were almost completely eluted after exposure at 37° C. to lymph fluid. The ability of lymph fluid to elute lysozyme from aluminum phosphate adjuvant did not change as the vaccine aged. Only 60% of the ovalbumin adsorbed to aluminum hydroxide was eluted during exposure to lymph fluid after the vaccine aged for 11 weeks at 4° C.
Iyer et al. (Vaccine 21 (2003) 1219-1223) discloses the finding that ovalbumin and dephoshorylated alpha casein were adsorbed in an aluminum hydroxide vaccine but were completely eluted when exposed to interstitial fluid. The vaccine nevertheless produced immunopotentiation compared to a solution of the protein. In contrast, alpha casein was completely adsorbed to aluminum hydroxide in both the vaccine and upon exposure to interstitial fluid. Immunopotentiation by aluminum hydroxide was also observed for alpha casein. The results indicated that antigen presenting cells can take up desorbed antigen from interstitial fluid as well as antigen adsorbed to aluminum-containing adjuvants.
Katz et al. (Journal of Virological Methods, 25 (1989) 101-108) discloses an ELISA for assessing the antigenic content of inactivated aluminum hydroxide adjuvanted virus vaccines. The ELISA is stated to supplement in vivo testing. Other in vitro methods comprise radioimmunoassay and methods requiring antigen desorption from aluminum hydroxide. Unlike such in vitro methods the ELISA did not suffer significant interference from the aluminum hydroxide except at high aluminium aluminum hydroxide concentrations. It is mentioned that previous attempts at an ELISA of intact vaccines suffered extreme inhibition by aluminum hydroxide. The article sets forth various explanations why the present ELISA works. In the ultimate paragraph it is suggested that the method may be applicable to aluminum hydroxide adjuvanted virus vaccines as a class.
Thraenhart et al. (Journal of Biological Standardisation, (1989) 17, 291-309) discloses an ELISA for in vitro potency testing of rabies virus vaccine by determination of rabies virus glycoprotein. The influence of aluminum hydroxide on the potency measurement was investigated and it was found that there was no influence.
U.S. Pat. No. 4,127,385 (Weeke) describes a method comprising mixing allergen extracts of horsehair and scale adhered to an alhydrogel with serum from an allergic patient to bind the free IgE of the serum to the allergen of the alhydrogel and subsequently adding radiolabelled anti-IgE and measuring the radioactivity. It is indicated that the method can be used to determine the strength or the storage life of allergen extracts adhered to alhydrogel. This type of prior art immunoassay has the disadvantage that antibodies not specific for the allergen will to a certain extent bind to the alhydrogel-allergen and result in an incorrect measurement.
The nature of molecular antigen adsorption to the solid phase carriers is very complex and largely unknown, and also it varies among different antigens depending on the chemical and structural nature of the antigen. Furthermore, the influence of the solid phase carrier in the reaction between antigen-specific IgE and antigen bound to a solid phase carrier is very complex and not fully known. Therefore, it has until now been believed that it is not possible to measure in vitro the immunological activity, including the allergenic activity and potential for inducing allergic reactions, of ready-to-use solid phase carrier vaccines comprising molecular antigens, or at least that it is not possible to measure it accurately. Thus, up to now it has been common practise practice to evaluate the immunological activity of a vaccine in vitro on the basis of a measurement of the immunological activity of the solution of molecular antigen used for the preparation of the ready-to-use solid phase carrier vaccine. The object of the present invention is to provide an in vitro method of evaluating the immunological activity of ready-to-use, solid phase carrier, molecular antigen vaccines.